DiBiase, C. | Search bioRxiv

bioRxiv2025

A Single-Cell Atlas of Transcriptional and Immunoglobulin Repertoire Evolution in Early B Cell Development

The development of human B cells in the bone marrow can be separated into functionally and transcriptionally distinct subsets, which while canonically described have yet to be profiled deeply and individually and with NGS and bioinformatic techniques. In this study, single-cell RNA sequencing was performed on 65,110 B cells from six healthy donors to observe the foundation of the diversity observed in mature immune repertoires. Following the established narrative of development, committed B cells undergo heavy and light chain recombination, which together form the functional BCR. However, a granular per-subset phenotypic analysis reveals proliferative bursts following each recombination event, an aspect of BCR recombination previously undescribed in the pro-B phase of development. Heavy and light chain pairing becomes more similar to that of mature, circulating B cells with progress through lymphopoiesis, deleting features associated with autoreactivity. Prominent among these repertoire alterations is a substantial shortening of heavy chain CDR3s, and changes in V, D, and J gene usage. This study provides a detailed snapshot of the transcriptomic and repertoire genetics landscape of developing B cells, providing a deeper understanding of the repertoire-shaping influence of early selection processes, and introducing new avenues for repertoire development research.

immunology↗

bioRxiv2024

Conformational ensemble-based framework enables rapid development of Lassa virus vaccine candidates

Lassa virus (LASV), an arenavirus endemic to West Africa, poses a significant public health threat due to its high pathogenicity and expanding geographic risk zone. LASV glycoprotein complex (GPC) is the only known target of neutralizing antibodies, but its inherent metastability and conformational flexibility have hindered the development of GPC-based vaccines. We employed a variant of AlphaFold2 (AF2), called subsampled AF2, to generate diverse structures of LASV GPC that capture an array of potential conformational states using MSA subsampling and dropout layers. Conformational ensembles identified several metamorphic domains--areas of significant conformational flexibility--that could be targeted to stabilize the GPC in its immunogenic prefusion state. ProteinMPNN was then used to redesign GPC sequences to minimize the mobility of metamorphic domains. These redesigned sequences were further filtered using subsampled AF2, leading to the identification of promising GPC variants for further testing. A small library of redesigned GPC sequences was experimentally validated and showed significantly increased protein yields compared to controls. Antigenic profiles indicated these variants preserved essential epitopes for effective immune response, suggesting their potential for broad protective efficacy. Our results demonstrate that AI-driven approaches can predict the conformational landscape of complex pathogens. This knowledge can be used to stabilize viral proteins, such as LASV GPC, in their prefusion conformation, optimizing them for stability and expression, and offering a streamlined framework for vaccine design. Our deep learning / machine learning enabled framework contributes to global efforts to combat LASV and has broader implications for vaccine design and pandemic preparedness.

immunology↗